Eukaryotic parasites of the unicellular type (a.k.a. protozoan or protist parasites) plague tens of millions of humans, often in tropical areas but notably also in developed countries. They mediate an enormous toll on health and life itself. Drugs to control these infections are limited and often address the acute rather than chronic phases of the infections which typically last for life. Attention to the development of new treatments is urgently needed. Two of the best-known and most studied groups are the kinetoplastids (including trypanosomatids) and apicomplexans, and an extensive literature implicates glycosylation-related processes in their biology, virulence and persistence. With the genomics revolution, and with fresh and elegant methods of genetic manipulation such as CRISPR/Cas9 gene editing, it is now possible to plan approaches to incisively and rigorously test the proposed roles of glycans genetically. Nevertheless, the technology is very new, and glycobiology is a specialty area of knowledge that is imposing to non- afficionados. What is needed at this moment in time is a resource that i) encapsulates a genomics organization of glycogenes relevant to the assembly of the classes and novel types of glycans produced by the various parasites, ii) provides an easy-to-implement recipe for disrupting these glycogenes, and, iii) for a representative set of examples, documents the feasibility of disruption and consequences on the cellular glycome. We will address this need in 3 specific aims that focus initially on one representative trypanosome, Trypanosoma cruzi, and one representative apicomplexan, Toxoplasma gondii. We have assembled a team of three experts, each at the University of Georgia, to ensure the best progress. The first aim will be led by Dr. Rick Tarleton, an expert on the genomics and immunobiology of T. cruzi, but not a glycobiologist. His group will lead the development of the new CRISPR/Cas9 gene editing method for T. cruzi and T. gondii, and implement the method in T. cruzi for a prioritized glycogene (glycan biosynthesis) list. The second aim will be led by Dr. Chris West, a glycobiologist who will help develop the overall glycogene hierarchies to be assessed in T. cruzi and T. gondii, and implement the CRISPR/Cas9 system for select T. gondii glycogenes. The third aim will be led by Dr. Lance Wells, an expert in glycomics, to assess glycosylation consequences in representative modified parasites. He will supervise B. Weatherly, M.S., a bioinformaticist who will assist in developing the glycogene hierarchy and algorithms for interpreting the mass spectrometry data in a manner suitable for dissemination. With this toolbox in hand, we envision that non-specialists in the fiel will be suitably prepared to make connections between their favorite areas of interest and the glycan components of the molecules that mediate those functions. In the future, we hope to expand this project as a U01 to generalize our tools to other major health threats such as T. brucei and various Leishmania spp. in the kinetoplastids, Cryptosporidium and Plasmodium (malaria) in the apicomplexans, and other important protozoan groups.